The sensation of pain can be triggered by any number of physical or chemical stimuli. In mammals, the peripheral terminals of a group of specialized small diameter sensory neurons, termed “nociceptors” mediate this response to a potentially harmful stimulus.
In efforts to discover better analgesics for the treatment of both acute and chronic pain, and to develop treatments for various neuropathic pain states, considerable research has been focused on the molecular mechanism of nociception. The response to heat, low extracellular pH, or capsaicin (the compound responsible for the hotness of hot peppers) is characterized by the persistent activation of nociceptors (Bevan and Gepetti, 1994, and Kress and Reeh, 1996). It has been shown that both heat and capsaicin are capable of activating dorsal root ganglion and trigeminal ganglion neurons via and influx of cations (Oh, et al. 1996, Kirshstein, et al. 1997). Additionally, moderately acidic conditions produce this response (Zeilhofer, et al., 1997) and also potentiate the response of nociceptors to heat and capsaicin (Kress, et al., 1996).
Capsaicin responses in isolated sensory neurons show dose-dependence and are also evoked by structural analogues of capsaicin (Szolcsanyi and Jancso-Gabor, 1975 and 1976). Resiniferatoxin (RTX), a natural product of Euphorbia plants is a particularly potent activator of the capsaicin response (Szallasi and Blumberg, 1989). Capsaicin and resiniferatoxin share a common vanilloid moiety, thus the capsaicin receptor is also termed the vanilloid receptor (VR). The capsaicin response is competitively inhibited by another structural analog, capsazepine (Bevan, et al., 1992) and by the non-selective cation channel blocker ruthenium red (Wood, 1988).
It was initially postulated that the VR is a non-selective cation channel with a preference for calcium. Consequently, a 45Ca2+-uptake assay using intact rat dorsal root ganglion (DRG) neurons has been used extensively to characterize structure-activity relations for vanilloids. Specific binding of [3H]RTX provided the first unequivocal proof for the existence of a VR and has furnished a new, biochemical tool to study VR pharmacology. Such studies, however, have been limited by the lack of availability of cloned VR species and sub-types, by the low levels of VR produced by the few cell types that naturally express such receptors in vivo, and by the limited expression levels heretofore obtained using transient recombinant expression technologies.
Interest in characterizing VRs led to the cloning of a functional rat capsaicin receptor (VR1), from a rat dorsal root ganglion cDNA library (Caterina, et al., 1997). The cDNA for the rat capsaicin receptor VR1 encodes an 838 amino acid protein (SEQ ID NO:9) with a predicted molecular mass of 95,000 Daltons.
Sequence analysis suggests that the receptor is composed of a 432 amino acid hydrophilic amino terminus that contains a proline-rich region followed by three ankyrin repeat domains, a membrane bound region that includes 6 beta-sheet transmembrane domains as well as an additional membrane-associated region between transmembrane segments 5 and 6, and a 154 amino acid carboxy terminus.
VR1 is activated not only by vanilloids but also by noxious heat and low pH. As predicted, this VR1 is a relatively non-selective cation channel with a preference for calcium. In Xenopus oocytes expressing VR1, vanilloids evoke inward currents, with RTX being approximately 20-fold more potent (EC50=39 nM) than capsaicin (EC50=710 μM). In VR1-transfected mammalian (HEK293) cells, capsaicin induces whole-cell currents with a potency of 110 nM. Taken together, these results suggest that VR1 corresponds to the site in DRG neurons that mediates calcium uptake.
Homology searches comparing the cloned rat capsaicin receptor VR1 to other known ligand gated channels have revealed some related receptors. The most highly homologous protein identified to date is the recently identified rat vanilloid-receptor-like protein 1 (VRL-1) (Caterina, et al. 1999). This protein shares approximately 49% identical amino acid residues and overall is 66% similar in sequence to the rat capsaicin receptor, VR1, and is predicted to have a tertiary structure quite similar to that of the capsaicin receptor. While the VRL-1 protein has been reported to respond to high temperatures by allowing an influx of cations, it is not a capsaicin receptor, as it is insensitive to capsaicin and capsaicin analogues.
Another class of receptors that shows some homology to the capsaicin receptor is the TRP (transient release potential) family of putative store-operated calcium channels. (Caterina, et al., 1997) also known as “trp channels”. Members of this family of receptors mediate the entry of extracellular Ca2+ in response to the depletion of intracellular Ca2+ stores (Clapham, 1996). The capsaicin receptor, while mediating the entry of Ca2+ and other cations in response to heat, low extracellular pH and capsaicin and related compounds, does not act as a store-operated calcium channel.
If vanilloid binding and calcium uptake are always mediated by the same receptor, a logical prediction would be that ligands mediating these two responses should display similar structure-activity relationships. With regard to DRG neurons expressing native VRs this is clearly not the case: structure-activity analysis of different vanilloid derivatives revealed that the various compounds have distinct potencies for receptor binding and for inducing 45Ca2+-uptake in rat DRG neurons. Although some compounds, such as RTX-amide, bind to VRs and evoke calcium influx with similar potencies, other vanilloids show relative selectivity for one or the other response. RTX represents one extreme. It is approximately 25-fold more potent for binding (using intact rat DRG neurons the Kd was reported to be 40 pM) than for inducing calcium uptake (EC50=1.0 nM). Capsaicin represents the opposite extreme. It evokes calcium influx with an EC50 of 270 nM but inhibits [3H]RTX binding with a 10-fold lower affinity of 3 uM. The most straightforward explanation appeared to be that RTX binding and calcium uptake detected two distinct classes of VRs. These putative receptors were referred to as R-type (preferentially labeled by RTX) and C-type (displaying a higher potency for capsaicin) VRs, respectively. This model was further supported by the identification of non-neuronal cell lines that exhibited calcium uptake in response to vanilloid stimulation (implying the presence of C-type VRs) but which lacked detectable RTX-specific binding sites.